Cloning of human anti-GM1 antibodies from motor neuropathy patients

Patients with multifocal motor neuropathy frequently have elevated titers of serum antibodies reactive with GM1 ganglioside. Although these antibodies may cause the syndrome, this has yet to be proven directly. As part of our studies on the nature and pathogenic potential of anti-GM1 antibodies, we have cloned B cells from the peripheral blood of 3 patients with multifocal motor neuropathy and generated four stable heterohybridoma cell lines secreting human monoclonal IgM anti-GM1 antibodies. In this report we describe the basic properties of these monoclonal antibodies in comparison with the patient's sera from which they were derived. The antibodies all differ in their pattern of reactivity with GM1 and other Gal(β1–3)GalNAc-containing glycoconjugates. They have widely varying thermal ranges and their reactivities are strongly influenced by the presence of accessory lipids. Affinity purification of the patient's sera with GM1 led to the identification of previously unrecognized paraproteins that were resolvable above the background of polyclonal anti-GM1 IgM. Our data demonstrate considerable heterogeneity in the immune response to GM1 both within individual sera and between different patients, which is likely to be of importance to their role in disease pathogenesis.     

Prenatal development of the human spinal cord. I. Ventral motor neurons

Differentiation of the ventral motor neurons were followed in the developing human spinal cord from week 8 to week 26 of intrauterine life by thionin staining and the rapid Golgi method. Ventral roots were seen as translucent rootlets by week 8 and the ventral motor neurons were clearly identifiable by week 10, indicating that the ventral grey was earlier to differentiate than the dorsal grey, which showed a small, darkly stained, undifferentiated cell population. Lateral groupings of the motor neurons in the cervical and lumbar enlargements were obvious by week 10. Nissl substance and dendrites had reached an adult pattern by about week 18 of intrauterine life.     

Serum IgM anti-GM1 ganglioside antibodies in lower motor neuron syndromes

Lower motor neuron syndromes (LMNS) are heterogenous conditions, which include patients with progressive lower motor neuron disease (LMND) and cases with the clinical phenotype of motor neuropathy (MN). The aim of this study was to estimate the IgM anti-GM1 ganglioside antibodies titer and the ratio of the light chains in order to define the presence of autoimmunity process in particular cases with LMNS. Twenty-eight patients were diagnosed with LMND and 15 patients were diagnosed with MN (10 patients with multifocal motor neuropathy with conduction block, five patients with MN without conduction block). Total of 103 patients with classical amyotrophic lateral sclerosis (ALS) and 50 healthy, age-matched persons were also tested. The IgM anti-GM1 ganglioside titer and the ratio of lambda/kappa light chains in serum were determined using the ELISA technique. High titer of IgM anti-GM1 antibodies were detected in serum of 46% LMND patients, 80% of MN patients, and 18% of the classical ALS cases. An elevated ratio of lambda/kappa light chains appeared in 18% of LMND patients, and in 67% of the MN cases. The lambda/kappa light chains ratio was normal in all ALS patients. The presence of elevated titer of IgM anti-GM1 ganglioside antibodies and the changed ratio of the light chains supports the presence of autoimmune process in LMNS and may provide clues for their management.     

A case of distal lower motor neuron syndrome associated with IgM anti-GM1 antibodies

A 34-year-old man had noted progressive weakness in his right hand. On admission at age 39, cranial nerves were not involved. Fasciculations were observed in his upper limb girdles. Neurological examination revealed severe wasting and weakness of arms and the right hand, whereas mild in the left hand. The deep tendon reflexes were absent in the upper extremities, but normal in the lower extremities. No sensory disturbances were observed. Motor and sensory nerve conduction velocities were normal, and multifocal conduction block was not observed. EMG showed neuropathic changes in all 4 limbs and sternocleidomastoideus muscles. Serum immunoelectrophoresis failed to detect an M protein. High-performance thin-layer chromatography with immunostaining revealed that his serum IgM reacted with GM1, but not reacted with GM2, GD1a, GD1b, and asialo-GM1.     

Conduction abnormalities induced by sera of patients with multifocal motor neuropathy and anti-GM1 antibodies

Increased titers of anti-GM1 antibodies have been associated with motor neuron disease and motor neuropathy with or without conduction block. To investigate the pathogenetic role of anti-GM1 antibodies we injected into rat tibial nerves sera from patients with multifocal motor neuropathy and conduction block (MMN) or progressive spinal muscular atrophy (PMA), both presenting anti-GM1 antibodies. Sera of patients with MMN produced reduction of amplitude and dispersion of compound muscle action potential from proximal stimulation. Morphometry revealed demyelination in 6.2% of fibers. Sera of patients with PMA did not produce clear-cut electrophysiological or morphological changes. Differential effects of sera from patients presenting high-titer anti-GM1 antibodies, but with distinct clinical syndromes, might depend on differences in anti-GM1 antibody affinity, valency, or ability to fix complement. Alternatively, circulating factors other than, or in addition to, anti-GM1 antibodies present in sera of patients with MMN, but not of PMA patients, might be responsible for conduction abnormalities and reproduce them after passive transfer. © 1993 John Wiley & Sons, Inc.     

Results of testing for anti-GM1 antibodies.

We used an ELISA technique to measure IgG and IgM antibodies to the ganglioside GM1, with the results expressed in arbitrary units. We tested 1007 sera from patients with peripheral neuropathy or muscle weakness. For IgG and IgM antibodies, the distribution of results differed significantly from a normal distribution. In the patient group, 81 of 1007 sera had elevated levels of IgG antibodies (> 10 units). Of these, 11 patients had very high levels (> 50 units). These 11 patients had diagnoses of GBS (4), motor neurone disease (3) or non-specific idiopathic neuropathy (4). For IgM antibodies, 115 of 1007 sera were positive (> 20 units). Of these, 18 patients had very high levels (> 50 units). These 18 patients had diagnoses of Guillain-Barré syndrome or Miller Fisher syndrome (4), multifocal motor neuropathy (4), motor neurone disease (2), non-specific neuropathy (2). We conclude that anti-GM1 antibodies in high titre are uncommon. Patients with multifocal motor neuropathy have high levels of antibody. However, patients with other disorders may also have high levels, so that anti-GM1 antibody levels alone are not a specific test for multifocal motor neuropathy. We found that antibodies to GM1 were present in the sera of patients with chronic idiopathic neuropathy, leading us to suggest that these antibodies may sometimes arise as a secondary response to disease.     

Regional distribution of cholinergic neurons in human spinal cord transections in the patients with and without motor neurons disease

Regional distribution of enzymic activities in acetylcholine (ACh) metabolism was examined on thinly-sectioned transverse slices of human spinal cords obtained during autopsy of 5 motor neuron disease (MND) and 5 control patients without MND. Choline acetyltransferase (ChAT) activity was highly concentrated in the ventral horn regions (gray and white matters) of cervical, thoracic and lumbar spinal cord of non-MND patients. This enzyme activity was found to be remarkably low in the ventral gray and white matter of MND patients compared with that of the controls. Although the distribution of acetylcholinesterase (AChE) activity was found to be high in both ventral and dorsal gray matter of the spinal cord, little difference was observed between each corresponding region of MND and control patients, except relatively low enzyme activity in the cervical ventral horn region of MND patients. Muscarinic cholinergic receptors, examined as specific [³H]quinuclidinylbenzilate ([³H]QNB) binding, was also highly concentrated in the ventral and dorsal gray matter of the control spinal cord, and was strongly reduced in the ventral horn region of MND patients, indicating a quite similar distribution pattern of ChAT activity. These biochemical changes of cholinergic transmission system may be paralleled to the morphological degeneration of the spinal lower motor neurons in MND patients. Activity of 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase), a marker enzyme of central myelin structure, was evenly distributed throughout the whole spinal cord section, without regard to the gray and white matter, of both MND and control patients.     

Descending modulation of the reflex reactions of the spinal motor neurons in human spinal cord damage

Changes of H-reflexes amplitude and Ia-inhibition intensity before, during and after voluntary contractions of skeletal muscles were studied in healthy subjects and patients with midthoracic injury of the spinal cord. The voluntary actions induced facilitation of H-reflexes and suppression of Ia inhibition in healthy people and in patients with paraparesis. In patients with paraplegia these voluntary actions induced either the depression of H-reflexes and increase of Ia inhibition intensity or caused no effect on these reactions. A relation between reflex reaction changes and posttraumatic structural and functional disturbance of descending paths were discussed.     

Clinical relevance of anti-GM1 IgM antibodies.

Anti-GM1 IgM antibodies were found in 23% of 56 patients with motor neuron disease (MND), in 19% of 69 patients with neuropathy and in 7% of 107 controls with other neurological and non-neurological diseases. Most patients had anti-GM1 IgM titers of 1:80 or less, while slightly higher titers (up to 1:640) were found in 1 patient with MND and 2 with neuropathy, and very high titers (1:20,480) were restricted to a patient with MND and an IgMk M-protein reacting with GM1, GD1b and asialo-GM1. Anti-GM1 IgM antibodies may be a normal constituent of the human antibody repertoire but their frequency and, at times, their levels are higher in patients with MND or neuropathy. The possible pathogenetic role of these antibodies remain to be established.     

Mercury in human spinal motor neurons

Inorganic mercury has been proposed as a neurotoxin that could cause sporadic motor neuron disease (SMND). We were therefore interested to see if mercury could be detected in the upper and lower motor neurons of SMND patients, and if mercury accumulated within motor neurons during life. Paraffin sections of formalin-fixed spinal cord (22 control adults, 20 SMND adults, 25 infants) and frontal primary motor cortex (9 control adults, 18 SMND adults, 20 infants) were stained with silver nitrate autometallography to detect ionic mercury. Mercury was found in the spinal motor neurons of 36% of adult control cases and 45% of adult SMND cases, with no significant difference between groups. No mercury was seen in infant spinal motor neurons, or in any adult or infant corticomotoneurons. In conclusion, many humans appear to accumulate mercury in their spinal motor neurons by the time they are adults, but mercury does not appear to play a major role in the loss of upper or lower motor neurons in SMND. Received: 27 December 1997 / Revised, accepted: 12 May 1998     

Intravenous immunoglobulin treatment in lower motor neuron disease associated with highly raised anti-GM1 antibodies

The effect of intravenous immunoglobulin (IVIg) treatmentwas studied in five patients with lower motor neuron disease associated with highly raised anti-GM1 antibodies but without evidence of conduction block on neurophysiological examination. The patients received IVIg treatment (0.4 g/kg for five consecutive days) in an openstudy. Only one patient responded to IVIg treatment, which wasconfirmed in a double blind, placebo controlled study (two placebotreatments and two IVIg treatments in a randomised order). However,after six months of maintenance IVIg treatment (0.4 g/kg weekly) muscleweakness gradually deteriorated below pretreatment levels despitecontinued treatment. It is concluded that the presence of raisedanti-GM1 antibodies does not identify a subgroup of patients with lowermotor neuron disease who respond to IVIg treatment and although somepatients with lower motor neuron disease may initially respond, IVIgtreatment does not seem to be sufficient as long term treatment.

     

Changes in corticospinal facilitation of lower limb spinal motor neurons after spinal cord lesions.

The projections from the cortex to the motor neurons of lower limb muscles were examined in 33 normal subjects and 16 patients with incomplete spinal cord lesions. Corticospinal neurons were excited by transcranial magnetic stimulation and the effects on single spinal motor neurons determined from peristimulus time histograms (PSTHs) of single tibialis anterior (TA) and soleus (SOL) motor units. In normal subjects magnetic stimulation produced a short latency facilitation of TA motor units but had little or no effect on SOL motor units. In the patients with spinal cord lesions magnetic stimulation also produced facilitation of TA but not SOL motor units; however, the mean latency of the TA facilitation was significantly longer (by about 14 ms) in the patient group. The F wave latencies were normal in all patients tested, suggesting that central rather than peripheral conduction was slowed. The duration of the period of increased firing probability (in TA motor units) was also significantly longer in the patients with spinal cord lesions. These changes may reflect the slowing of conduction and dispersal of conduction velocities in the corticospinal pathways as a consequence of the spinal cord lesion. No significant correlations were found between the delay of the TA facilitation and the clinical deficits in this group of patients.     

Patterns of reactivity with anti-glycolipid antibodies in human primary brain tumors

Antibodies against carbohydrates of three glycolipids were used to determine patterns of immunohistochemical reactivity of histologically identifiable cell subpopulations in 101 human primary brain tumors. For all tumor types fibrillary cells, polar cells, and gemistocytes (commonly seen in astrocytomas and ependymomas) stained more frequently for galactosylcerebroside with mAbO1 than small tumor cells and macrophages. Frequency of staining for sulfatide with mAbO4 was fibrillary > polar > small cells = macrophages. Gemistocytes stained more frequently with mAbO4 than polar cells in all tumors except low grade astrocytomas. These data indicate that tumors classified on histological grounds as astrocytic are often stained with antibodies that recognize oligodendrocytes and their progenitors. Thus, anti-glycolipid antibodies used in the study of developmental lineage may offer useful tools for classification of human brain tumors. Staining of fibrillary cells, polar cells, and gemistocytes for paragloboside directly with mAb F1H11 was much less common than with mAbO1, but this increased by pretreatment of the tissues with neuraminidase (F1H11 + N). Of particular note was the finding that small tumor cells frequently stained with F1H11 + N. Evidence that these were not macrophages was obtained using double immunostaining with F1H11 + N and anti-macrophage antibodies. In astrocytomas the frequency of small tumor cells immunostained with F1H11 + N was high grade > anaplastic > low grade, demonstrating a correlation of this tumor cell population with more F1H11 + N may be of value in identifying small, ana-plastic tumor cells, especially in small biopsies or tissue taken adjacent to the main tumor mass. Copyright © 1994 Wiley-Liss, Inc.     

Reactivity of a human monoclonal anti-GM1 and anti-GD1b IgM antibody with human neurons in cultures

A serum containing a monoclonal IgM λ with anti-GM1 and anti-GD1b activity was obtained from a patient with upper motor neuron syndrome. By indirect immunocytochemical techniques with double staining, the patient's IgM strongly stained membranes of neurons in primary cultures of fetal central and peripheral nervous system. It was cytotoxic for neurons in two human neuroblastoma established cell lines in a complement-dependent chromium release assay. These results are in keeping with the hypothesis of a direct pathogenetic role of such monoclonal anti-GM1 and GD1b IgM antibodies.     

Selective vulnerability of spinal cord motor neurons to non-NMDA toxicity

We previously reported that alpha-motor neurons in organotypic cultures of rat spinal cord (OTC-SC) are resistant to excitotoxicity induced through NMDA receptors. Here we describe the effects of non-NMDA glutamate receptor agonists kainic acid (KA) and quisqualic acid (QUIS) on motor neurons in OTC-SC. Large ventral horn acetylcholinesterase-positive neurons (VHANs), most of which are motor neurons, were quite sensitive to QUIS and KA toxicity and displayed losses of 95% and 94%, respectively. Small VHANs were reduced by 41% and 61% only. Identical results were obtained in cultures stained for non-phosphorylated neurofilaments. These observations demonstrate that alpha-motor neurons are considerably more sensitive to KA and QUIS than to NMDA toxicity. The proposed excitotoxic mechanism of ALS, therefore, is most likely mediated through non-NMDA glutamate receptors.     

Cultured rat spinal cord neurons: interaction with motor neuron disease immunoglobulins

Conditions have been developed for the culture of rat spinal cord neurons in serum-free media supplemented with hormones and growth factors. Neurons were identified by immunofluorescence-labeled anti-neurofilament antibody, and their growth was monitored by assay of choline acetyltransferase and cholinesterase activities. Activities of these enzymes were considerably higher than those of comparable cultures in serum supplemented media in which there were visibly many more nonneuronal cells. Serum immunoglobulins from patients with motor neuron disease showed enhanced binding to rat spinal cord cells maintained in both serum-supplemented and serum-free media, as compared with those from normal healthy individuals. Enhanced binding was more marked with the latter cells, presumably because of the higher proportion of neuronal cells in these cultures. Serum immunoglobulins from patients with other neurologic disorders showed a similar binding to that of the normal controls. The results demonstrate the presence of an immune response to spinal cord cell membrane components in patients with motor neuron disease, although whether the response is primary or secondary in the disease process remains unclear.     

Normal human plasma contains antibodies that specifically block neuropathy-associated human anti-GM1 IgG-antibodies

Intravenous immunoglobulin (IVIg) is used in the treatment of a variety of autoimmune diseases. The blocking of disease-associated antibodies by anti-idiotype antibodies present in IVIg has been proposed as an action mechanism. Anti-GM1 antibodies have been implicated in motor neuropathies. Although IVIg is frequently applied for these diseases, the presence in IVIg or in human plasma of anti-idiotype antibodies that recognize anti-GM1 antibodies has not been clearly demonstrated. Here we present evidence that normal human plasma contains antibodies that inhibit the binding of anti-GM1 IgG-antibodies from neuropathy patients but do not inhibit anti-GM1 IgG-antibodies of rabbit origin with the same fine specificity. The significance of these findings in the course of acute and chronic neuropathies is discussed.     

IgG anti-GM1 antibodies from patients with acute motor neuropathy are predominantly of the IgG1 and IgG3 subclasses

Increased titers of IgG anti-GM1 and anti-asialo GM1 (GA1) ganglioside antibodies are present in some patients with the Guillain-Barré syndrome, particularly with the motor axonal variant, and following infection with Campylobacter jejuni or parenteral administration of gangliosides. The subclass distribution of IgG anti-GM1 or GA1 antibodies from 19 patients with acute motor neuropathy and elevated antibody titers were measured by ELISA using mouse monoclonal antibodies specific for human IgG subclasses. The anti-GM1 or GA1 antibodies were predominantly of the IgG1 and IgG3 subclasses, which are capable of complement fixation, and are characteristic of a T cell-dependent antibody response.